Antibiotic Prescription in Veterinary Consultations in Bhutan: A Retrospective Cross-Sectional Study.

The veterinary prescription of antibiotics in low- and middle-income countries (LMIC) remains largely undocumented. In Bhutan, however, the national veterinary service keeps records of their activities and prescriptions, which offer an opportunity to establish a benchmark to assess the use of these agents in this and other LMIC. A cross-sectional retrospective study was designed and 2,266 handwritten veterinary records from 2017 were sampled from 23 animal health premises (AHPs) to estimate individual and an overall proportion of consultations that resulted in an antibiotic prescription. The frequency of antibiotic prescription per species, type of AHP, and according to WHO's AWaRe index and OIE list of priority antimicrobials were also explored. It was estimated that 31% (95% confidence interval: 29-33%; intracluster correlation: 0.03) of the veterinary consultations resulted in an antibiotic prescription. The incidence of antibiotic prescription was highest in consultations of poultry across AHP. Across species, diarrhea and wounds were frequently treated with broad-spectrum antibiotics including sulfonamides, tetracyclines, trimethoprim + sulfa, and penicillin. Between 45% and 70% antibiotics prescribed correspond to AWaRe's access group and up to 25% to AWaRe's watch group. Over 70% of antibiotics dispensed in veterinary consultations for any species correspond to the OIE's veterinary critically important antimicrobial agents. Overall, the study demonstrated positive features of veterinary antimicrobial stewardship in Bhutan, given the conservative proportion of consultation that results in this type of prescription and the type of antibiotic prescribed. Although the veterinary service closely follows the Bhutanese Standard Treatment Guidelines, the prescription of antibiotics to key species should be closely monitored. Our study suggests that further improvements of antibiotic stewardship can be achieved through standardisation of antibiotic prescription to some species, a revision of the guidelines toward reducing the prescription of antibiotics of high relevance for human medicine, and by including details of clinical investigation, use of tests, and treatment outcomes in veterinary consultation records.

Cerebral cysticercosis in a wild Bengal tiger (Panthera tigris tigris) in Bhutan: A first report in non-domestic felids.

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population.

Seroprevalence and risk factors of canine distemper virus in the pet and stray dogs in Haa, western Bhutan.

BACKGROUND: Canine Distemper Virus (CDV) is a highly contagious virus belonging to family Paramyxovirade, genus Morbillivirus and responsible for high morbidity and mortality in dogs worldwide. Infected domestic dogs can cause spillover infections to wild carnivores that are in contact. We conducted a seroprevalence survey of CDV in domestic dogs in two areas of western Bhutan (Haa district) located at the periphery of the Jigme Khesar Strict Nature Reserve, which is home to several endangered wildlife. A total of 238 serum samples, 119 each from the pet and stray dog, were collected during summer and winter seasons. Samples were tested for CDV antibodies using a sandwich enzyme-linked immune-sorbent assay (ELISA) test. RESULTS: The seroprevalence of CDV was found to be 11.3% (95% CI 6.7-14.2). Dogs sampled during winter were less likely to test seropositive against CDV antibodies than those sampled during summer (adjusted odds ratio: -2.6; 95% CI: - 1.2-6.1). Dogs in good body condition were found to be more likely to test seropositive against CDV than dogs in poor condition and obese dogs (adjusted odds ratio: 2.2; 95% CI: 0.1-5.9). There were no significant differences in the seroprevalence of CDV among different sexes, breeds and age classes, pet and stray dogs and between the two study sites. CONCLUSIONS: Our study indicates that CDV seroprevalence was equally distributed among pet and stray dogs. We suggest strengthening the management practices of dogs through responsible dog ownership, dog population management and waste management to minimize the transmission risk of infectious diseases to wildlife.

Revisiting an old disease? Risk factors for bovine enzootic haematuria in the Kingdom of Bhutan.

Bovine enzootic haematuria (BEH) is a debilitating disease of cattle caused by chronic ingestion of bracken fern. Control of BEH is difficult when bracken fern is abundant and fodder resources are limited. To fill a significant knowledge gap on modifiable risk factors for BEH, we conducted a case-control study to identify cattle management practices associated with BEH in the Bhutanese cattle population. A case-control study involving 16 of the 20 districts of Bhutan was carried out between March 2012 and June 2014. In Bhutan sodium acid phosphate and hexamine (SAP&H) is used to treat BEH-affected cattle. All cattle greater than three years of age and treated with SAP&H in 2011 were identified from treatment records held by animal health offices. Households with at least one SAP&H-treated cattle were defined as probable cases. Probable case households were visited and re-classified as confirmed case households if the BEH status of cattle was confirmed following clinical examination and urinalysis. Two control households were selected from the same village as the case household. Households were eligible to be controls if: (1) householders reported that none of their cattle had shown red urine during the previous five years, and (2) haematuria was absent in a randomly selected animal from the herd following clinical examination. Details of cattle management practices were elicited from case and control householders using a questionnaire. A conditional logistic regression model was used to quantify the association between exposures of interest and household BEH status. A total of 183 cases and 345 controls were eligible for analysis. After adjusting for known confounders, the odds of free-grazing for two and three months in the spring were 3.81 (95% CI 1.27-11.7) and 2.28 (95% CI 1.15-4.53) times greater, respectively, in case households compared to controls. The odds of using fresh fern and dry fern as bedding in the warmer months were 2.05 (95% CI 1.03-4.10) and 2.08 (95% CI 0.88-4.90) times greater, respectively, in cases compared to controls. This study identified two husbandry practices that could be modified to reduce the risk of BEH in Bhutanese cattle. Avoiding the use of bracken fern as bedding is desirable, however, if fern is the only available material, it should be harvested during the colder months of the year. Improving access to alternative fodder crops will reduce the need for householders to rely on free-grazing as the main source of metabolisable energy for cattle during the spring.

A cross-sectional survey of population demographics, the prevalence of major disease conditions and reason-specific proportional mortality of domestic cattle in the Kingdom of Bhutan.

We describe the population demographics, rudimentary measures of reproductive performance, the prevalence of major disease conditions and reason-specific proportional mortality for cattle owned by villagers in 16 dzongkhags (districts) in Bhutan based on the findings of a cross-sectional study carried out between March 2012 and May 2014. The animal health issues that were of concern for livestock owners are also identified. Study households were selected using a stratified, two-stage cluster design. Districts (dzongkhags) formed the strata. Villages within dzongkhags were the first sampling stage and households within villages the second sampling stage. All cattle within each selected household comprised the study population. Questionnaires were collected from 409 households with 1480 standing cattle. A total of 71.0% (95% CI 67.9%-74.1%) of the standing cattle population were female and the median age of cattle was 5 years (Q25 3 years; Q75 7 years). Exotic breeds of cattle (Jerseys, Brown Swiss, and their crosses) comprised 41.4% of the cattle population with local breeds making up the remainder. Although exotic breeds of cattle had a significantly lower age at first calving (median 4 years) compared to local breeds (median 4.8 years) there was no significant difference in the number of calving events per cow for the two breed groups. Diarrhoea was the most prevalent disease condition with 2.8 (95% CI 1.5-4.6) cases per 100 animals followed by bovine enzootic haematuria with 1.9 (95% CI 1.0-3.3) cases per 100 animals. The most frequently cited cause of death was misadventure (proportional mortality 26.2%, 95% CI 15.7%-39.2%) followed by old age (17.8%, 95% CI 9.5%-29.4%). A lack of access to adequate fodder and pasture was the animal health issue that was cited by interviewees most frequently. We provide no evidence that exotic breeds of cattle have superior reproductive performance compared with local breeds. The major cattle health concern cited by interviewees, lack of access to fodder, is likely to contribute to suboptimal productivity and the relatively high incidence of bovine enzootic haematuria in this population. We propose that a prospective cohort study of Bhutanese cattle is warranted to more fully elucidate factors affecting the productivity and longevity of cattle in Bhutan.

Size and demography pattern of the domestic dog population in Bhutan: Implications for dog population management and disease control.

Understanding the demography of domestic dogs is essential to plan the dog population management and rabies control program. In this study, we estimated the owned and stray dog population and the proportion of owned dogs that are free-roaming in Bhutan. For this, a cross-sectional household surveys were conducted in six districts (both urban and rural areas) and two border towns in southern Bhutan. The population estimation was done by extrapolation of the mean number of dogs per household and dogs per person, whilst mark-resight survey was conducted to estimate the proportion of owned dogs that were free-roaming. A total of 1,301 (rural:585; urban:716) respondents (one per household) were interviewed of which 173 households (24.4%) in urban areas owned 237 dogs whilst 238 households (40.8%) in rural areas owned 353 dogs. The mean number of dogs per dog owning household was estimated to be 1.44 (urban:1.37 dogs; rural:1.48 dogs) and dogs per household was estimated to be 0.45 (urban:0.33; rural:0.60). The dog: human ratio was 1:16.30 (0.06 dogs per person) in urban areas and 1:8.43 (0.12 dogs per person) in rural areas. The total owned dog population based on the mean number of dogs per household and dogs per person were estimated to be 65,312 and 71,245 in the country, respectively. The male: female ratio of the owned dog was 1.31:1 in urban areas and 2.05:1 in rural areas. Majority of the dogs were local non-descript breeds in both urban (60.8%) and rural (78%) areas, and the most common source was acquisition from friends or family (44.7%). The stray dog population in Bhutan was estimated to be 48,379 (urban:22,772; rural:25,607). Of the total estimated owned dog population in the two border towns, the proportion that were found free-roaming was estimated to be 31%. The different dog population estimation methods were compared and discussed in this paper. This study generated baseline data on the demographic patterns of the owned and stray dogs in Bhutan which will be useful for planning and monitoring dog population management and rabies control program in the country.

Comparison of mark-resight methods to estimate abundance and rabies vaccination coverage of free-roaming dogs in two urban areas of south Bhutan.

In Bhutan, Capture-Neuter-Vaccinate-Release (CNVR) programs have been implemented to manage the dog population and control rabies, but no detailed evaluation has been done to assess their coverage and impact. We compared estimates of the dog population using three analytical methods: Lincoln-Petersen index, the Chapman estimate, and the logit-normal mixed effects model, and a varying number of count periods at different times of the day to recommend a protocol for applying the mark-resight framework to estimate free-roaming dog population abundance. We assessed the coverage of the CNVR program by estimating the proportion of dogs that were ear-notched and visually scored the health and skin condition of free-roaming dogs in Gelephu and Phuentsholing towns in south Bhutan, bordering India, in September-October 2012. The estimated free-roaming dog population in Gelephu using the Lincoln-Petersen index and Chapman estimates ranged from 612 to 672 and 614 to 671, respectively, while the logit-normal mixed effects model estimate based on the combined two count events was 641 (95% CI: 603-682). In Phuentsholing the Lincoln-Petersen index and Chapman estimates ranged from 525 to 583 and 524 to 582, respectively, while the logit-normal mixed effects model estimate based on the combined four count events was 555 (95% CI: 526-587). The total number of dogs counted was significantly associated with the time of day (AM versus PM; P=0.007), with a 17% improvement in dog sightings during the morning counting events. We recommend to conduct a morning marking followed by one count event the next morning and estimate population size by applying the Lincoln-Peterson corrected Chapman method or conduct two morning count events and apply the logit-normal mixed model to estimate population size. The estimated proportion of vaccinated free-roaming dogs was 56% (95% CI: 52-61%) and 58% (95% CI: 53-62%) in Gelephu and Phuentsholing, respectively. Given coverage in many neighbourhoods was below the recommended threshold of 70%, we recommend conducting an annual "mass dog vaccination only" campaign in southern Bhutan to create an immune buffer in this high rabies-risk area. The male-to-female dog ratio was 1.34:1 in Gelephu and 1.27:1 in Pheuntsholing. Population size estimates using mark-resight surveys has provided useful baseline data for understanding the population dynamics of dogs at the study sites. Mark-resight surveys provide useful information for designing and managing the logistics of dog vaccination or CNVR programs, assessing vaccination coverage, and for evaluating the impact of neutering programs on the size and structure of dog populations over time.

Evaluation of a monoclonal antibody-based rapid immunochromatographic test for direct detection of rabies virus in the brain of humans and animals.

Rabies diagnosis uses a direct fluorescent antibody test (FAT) that is difficult, costly, and time-consuming, and requires trained personnel. We developed a rapid immunochromatographic test (RICT) for the diagnosis of rabies. The efficacy of the RICT was compared with that of the FAT. Brain samples were collected from humans, dogs, cats, and other animals in Sri Lanka (n = 248), Bhutan (n = 27), and Thailand (n = 228). The sensitivity (0.74-0.95), specificity (0.98-1.0), positive predictive value (0.98-1.0), negative predictive value (0.75-0.97), accuracy (0.91-0.98), and kappa measure of agreement (0.79-0.93) were all satisfactory for animal samples and samples preserved in 50% glycerol saline solution. Because the RICT showed high sensitivity but low specificity with human brain samples, it is unsuitable for confirming rabies in humans. No amino acid substitutions were found in the antibody attachment sites of the nucleoprotein gene with FAT-positive, RICT-negative samples. The RICT is reliable, user friendly, rapid, robust, and can be used in laboratories with a modest infrastructure.